Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B from Geobacillus collagenovorans MO-1

In this work, Pz peptidase B, an intracellular M3 metallopeptidase that is found in the thermophile Geobacillus collagenovorans MO-1, was crystallized using the counter-diffusion method.

Abstract

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as ‘Kibo’) at the International Space Station (ISS). The crystal belonged to the trigonal space group P3₁21, with unit-cell parameters a = b = 87.64, c = 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

1. Introduction  

The thermophile Geobacillus collagenovorans MO-1, which can degrade collagens, was isolated from soil. Several characteristic enzymes related to collagen degradation were found in this strain and have been extensively studied (Okamoto et al., 2001 ▶; Miyake et al., 2005 ▶; Itoi et al., 2006 ▶). Two intracellular M3 metallopeptidases, Pz peptidases A and B, are critical in hydrolyzing collagen-derived peptides by recognizing a collagen-specific tripeptide unit, -Gly-Pro-X-, in this strain. Although the two enzymes show very low identity in their primary structure (22%), Pz peptidases A and B exhibit similar substrate specificity (Miyake et al., 2005 ▶). Very recently, the crystal structure of Pz peptidase A was determined at 1.80 Å resolution; one of the striking features that was revealed was a tunnel that functions as a gateway for substrate and reactant molecules, with the active site located in the centre of the globular protein molecule (Kawasaki et al., 2010 ▶). We are specifically interested in comparing the molecular structures of the two Pz peptidases and determining the catalytic mechanism of Pz peptidase B using X-ray crystallographic analysis. In contrast, the enzyme thimet oligopeptidase (TOP), a mammalian enzyme, has a similar substrate specificity to the Pz peptidases but shows a much lower identity (14% at most). In addition, there is only a small difference between the catalytic parameters of the Pz peptidases and of TOP (Sugihara et al., 2007 ▶; Dando et al., 1993 ▶). The structure of TOP has been reported not to contain a tunnel for substrates and reactants as in Pz peptidase A; rather, it contains a channel that runs the length of the entire protein molecule (Brown et al., 2001 ▶; Ray et al., 2004 ▶). Therefore, we were interested in identifying the differences in structure between Pz peptidase B, Pz peptidase A and TOP. Here, we report the overexpression, purification, crystallization and X-ray analysis of native and selenomethionyl (SeMet) Pz peptidase B.

2. Materials and methods  

2.1. Protein expression and purification of Pz peptidase B  

The gene for Pz peptidase B contained in a pUC119 plasmid was subcloned into a pET11b expression vector for overexpression (Miyake et al., 2005 ▶). The plasmid, named pOPB-1, was introduced into Escherichia coli strain BL21 (DE3). The transformant was grown in LB medium containing 50 µg ml⁻¹ ampicillin at 310 K. The purification method for Pz peptidase B has been described previously (Sugihara et al., 2007 ▶). Briefly, cells harvested from the culture were washed with saline and resuspended in 100 ml buffer I (50 mM Tris–HCl pH 7.5) after disintegration by sonication. The supernatant was collected after centrifuging the homogenate at 6000g for 10 min. The supernatant was kept at 333 K for 30 min and denatured particulates were removed by centrifugation at 10 000g for 10 min. The resulting supernatant was applied onto a DEAE-cellulose column (GE Healthcare) equilibrated with buffer I. Elution was performed with a linear gradient of buffer I containing 0–0.5 M NaCl. The eluted fractions containing Pz peptidase B were concentrated using an Amicon PM-10 membrane (Millipore) and the concentrate was further purified using a Sephacryl S-300 column (GE Healthcare) equilibrated with buffer I. The active flowthrough was pooled and concentrated. The product homogeneity was determined by SDS–PAGE and functional assessment of Pz peptidase B. A standard assay of Pz peptidase B activity in purification was performed using Pz-PLGPR (Bachem, Bubendorf, Switzerland) as reported previously (Miyake et al., 2005 ▶; Sugihara et al., 2007 ▶). One unit of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 µmol Pz-PLGPR in 1 min at 333 K.

2.2. Crystallization and data collection  

The purified Pz peptidase B was concentrated to 20 mg ml⁻¹ in 50 mM Tris–HCl pH 7.5. Initial crystallization screening was carried out with Crystal Screen, Crystal Screen 2 (Hampton Research) and Wizard 1, 2 and 3 (Emerald BioSystems) using both the hanging-drop and sitting-drop vapour-diffusion methods. 1 µl protein solution was mixed in a 1:1 ratio with each of the crystallization solutions and the resultant drops were incubated at 293 K. Dodecahedron-shaped crystals grew in drops containing 0.1 M bis-Tris–HCl pH 5.9, 2 M ammonium sulfate after 3 d. Subsequent refinement of the conditions gave optimal crystallization conditions consisting of 0.1 M bis-Tris–HCl pH 5.9, 2.3 M ammonium sulfate. Crystals obtained from drops prepared by mixing 2 µl protein solution and 2 µl reservoir solution grew to dimensions of 0.5 × 0.2 × 0.1 mm at 297 K within 5 d (Fig. 1 ▶). SeMet-substituted Pz peptidase B was crystallized using the same conditions as those optimized for native Pz peptidase B. Crystals of native and SeMet-substituted Pz peptidase B were flash-cooled using a solution consisting of 0.1 M bis-Tris–HCl pH 5.9, 2.3 M ammonium sulfate with 22%(v/v) 2-propanol as a cryoprotectant. X-­ray diffraction data were collected from native and SeMet-substituted protein crystals at 93 K using an ADSC Quantum 210r CCD detector on BL-­5A at the Photon Factory, High Energy Acceleration Research Organization, Tsukuba, Japan. The CCD detector was placed at a distance of 200 mm; an exposure time of 3 s allowed a 180° data set to be obtained (0.5° oscillation angle). Data were collected from a native crystal to a resolution of 2.2 Å and MAD data were collected from an SeMet-substituted crystal to resolutions of 2.2 Å (peak) to 2.4 Å (remote). Crystals suitable for high-resolution X-ray crystallographic analysis were obtained using a combination of the micro-seeding and counter-diffusion methods at the International Space Station (ISS). Seed crystals were obtained on Earth under the conditions described above and were transferred into 100 mM bis-Tris pH 5.9, 2.3 M ammonium sulfate. Space-grown crystals were obtained during the course of the Fourth Session of JAXA’s Microgravity Protein Crystallization Experiment (JAXA-PCG), which was performed using the Protein Crystallization Research Facility (PCRF) in the Japanese Experiment Module (JEM; also known as ‘Kibo’) at the ISS. The crystal was transferred to cryoprotectant consisting of 0.1 M bis-Tris–HCl pH 5.9, 2.3 M ammonium sulfate, 30%(v/v) glycerol. Several minutes later, it was scooped up in a cryoloop and flash-cooled in liquid nitrogen. It was then mounted on a goniometer in a nitrogen stream at 100 K. The X-­ray diffraction data were collected using a Rayonix MX-225HE CCD detector on BL44XU at SPring-8, Hyogo, Japan. A total of 360 images were collected at a crystal-to-detector distance of 160 mm with an oscillation angle of 0.5° and an exposure time of 5 s per image. Data were collected to a resolution of 1.6 Å. All data sets were indexed, integrated and scaled with the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶).

Figure 1.

A crystal of recombinant Pz peptidase B obtained using 0.1 M bis-Tris–HCl pH 5.9, 2.3 M ammonium sulfate on earth. The crystal dimensions are about 0.5 × 0.2 × 0.1 mm. The scale bar is 0.1 mm in length.

3. Results and discussion  

In the initial screening, cubic crystals with dimensions of approximately 50 × 50 × 50 µm were obtained within 3 d using the condition 50 mM Tris–HCl pH 9.0, 25 mM trimethylamine N-oxide, 12%(v/v) PEG 1000. However, further optimization of crystal growth under similar conditions yielded no crystals that diffracted to beyond 5.0 Å resolution. In contrast, X-ray diffraction data were collected from dodeca­hedron-shaped crystals of native Pz peptidase B grown on Earth and at the ISS to resolutions of 2.2 and 1.6 Å, respectively. The crystal grown at the ISS belonged to the trigonal space group P3₁21 or P3₂21, with unit-cell parameters a = b = 87.64, c = 210.5 Å. Assuming the presence of one molecule per asymmetric unit, the calculated Matthews coefficient (V _M) was 3.4 ų Da⁻¹ (Matthews, 1968 ▶); the solvent content of the crystal was calculated to be 63.8%. The high-resolution data set collected from crystals obtained in the JAXA-PCG project was used as a reference for scaling all native data sets and the combined data were used for phase determination and refinement. Crystals obtained in Kibo at the ISS were dodecahedron-shaped with dimensions of approximately 0.5 × 0.3 × 0.2 mm. The crystals obtained in space were of better quality (1.6 Å resolution) than those obtained on Earth (2.2 Å resolution) and are suitable for more precise structure analyses. The difference in resolution indicates that the microgravity in space yields crystals of high quality. Since gravity is one of the parameters controlling the quality of protein crystals, and buoyancy-driven convection and sedimentation are greatly reduced in space, the environment in space has been posited to be ideal for crystallization of Pz peptidase B. As a result, parameters other than gravity dominate the crystal-growth mechanism in space and hence provide a unique opportunity to study the crystal-growth process. SeMet-substituted Pz peptidase B crystals diffracted to beyond 2.4 Å resolution on BL-5A at the Photon Factory. In order to determine the structure using the MAD method, three wavelengths for data collection, 0.97919 Å (peak), 0.97900 Å (edge) and 0.96396 Å (remote), were selected on the basis of the X-­ray absorption spectrum at the Se K edge. Data-collection statistics for SeMet-substituted and native crystals are summarized in Table 1 ▶.

Table 1. Data-collection statistics for SeMet-substituted and native crystals.

Values in parentheses are for the highest resolution shell.

	SeMet			Native
Crystal	Peak	Edge	Remote	Grown on earth	Grown in space
Space group	P3121			P3121	P3121
Wavelength (Å)	0.97919	0.97900	0.96396	0.90000	0.90000
Unit-cell parameters (Å)	a = b = 87.99, c = 209.7			a = b = 88.15, c = 210.8	a = b = 87.64, c = 210.5
Observed reflections	256545	227873	202190	420046	964525
Unique reflections	92600	81376	71728	47527	146530
Resolution (Å)	50.0–2.20 (2.24–2.20)	50.0–2.30 (2.34–2.30)	50.0–2.40 (2.44–2.40)	50.0–2.20 (2.24–2.20)	40.0–1.60 (1.62–1.60)
R merge (%)	6.2 (36.5)	5.7 (34.6)	4.2 (23.5)	10.8 (25.8)	6.0 (41.1)
〈I/σ(I)〉	29.7 (3.4)	30.8 (4.0)	32.0 (4.4)	62.1 (15.1)	31.9 (2.7)
Multiplicity	2.8 (2.6)	2.8 (2.8)	2.9 (2.8)	8.9 (8.2)	6.6 (4.1)
Completeness (%)	98.9 (98.4)	99.1 (99.9)	99.1 (99.9)	96.4 (99.7)	97.0 (96.1)

MAD data in the resolution range 50.0–2.4 Å were used for phase determination. Nine selenium sites were found using the SOLVE program (Terwilliger & Berendzen, 1999 ▶). The space group was determined to be P3₁21 from an electron-density map using the initial phases. The initial phases were modified by solvent flattening using the RESOLVE program (Terwilliger, 2000 ▶) and were extended to 2.2 Å. The initial model was constructed automatically using the ARP/wARP program (Langer et al., 2008 ▶). On this occasion, a native data set at 1.6 Å resolution was combined with the phase output from RESOLVE and 5% of the reflections were selected for cross-validation. Data were collected to 1.6 Å resolution on BL44XU at SPring-8 because the long c axis of the crystals (c = 210.5 Å) prevented the collection of high-resolution data. Refinement of the model structure of Pz peptidase B is currently in progress. In addition, crystallization conditions for complexes of Pz peptidase B with the inhibitors PPI-1 and PPI-2 [PPI-1, benzyloxycarbonyl-(l,d)-Phe-(PO₂CH₂)-(l,d)-Ala-Lys-Ser; PPI-2, Gly-Pro-Phe-(PO₂CH₂)-Gly-Pro-Nle], which also inhibit Pz peptidase A (Kawasaki et al., 2007 ▶), are now being analyzed.